Key Words: Organophosphate esters; Gel permeation chromatograph; Solid phase extraction; Human serum; Gas chromatography-mass spectrometer

Transcription

1 CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 43, Issue 7, July 2015 Online English edition of the Chinese language journal Cite this article as: Chin J Anal Chem, 2015, 43(7), RESEARCH PAPER Determination of Organophosphate Esters in Human Serum Using Gel Permeation Chromatograph and Solid Phase Extraction Coupled with Gas Chromatography-Mass Spectrometry LI Peng 1, LI Qiu-Xu 1, MA Yu-Long 1, JIN Jun 1,2, *, WANG Ying 1, TIAN Yang 1 1 College of Life and Environment Sciences, Minzu University of China, Beijing 10081, China 2 Engineering Research Center of Food Environment and Public Health, Beijing 10081, China Abstract: A gel permeation chromatograph (GPC) coupled with solid phase extraction and gas chromatography-mass spectrometric (GPC-SPE-GC/MS) method was developed to analyze seven kinds of organophosphate esters, including Tri-n-butylphosphate, Tri(2-chloroethyl)phosphate, Triphenyl phosphate, Tris(2-butoxyethyl) phosphate, Tri-o-tolylphosphate, Tri-m-tolylphosphate and Tri-p-tolylphosphate in human serum. The recoveries of cleanup methods between GPC-silica/alumina column and H 2 SO 4 -silica/ sulfuric acid gel column were compared. The purification method with the GPC-silica/alumina column didn t destroy the structure of organophosphate esters (OPEs) and could effectively remove some proteins and lipid matrix influence in serum. The developed method was verified using the spiked blank and the spiked serum, and the good recoveries and reproducibilities were achieved. The recoveries of all of OPEs in spiked blank (n = 3) were all more than 75%. The recoveries of d 12 -TCEP and d 15 -TPhP in human serum samples (n = 9) were 86.3% ± 21.6% and 103.1% ± 16.5%, respectively. In human serum samples (n = 9), the detection ratios for TnBP, TCEP, TPhP, TBEP and m-ttp were more than 90% in all of the serum samples, p-ttp was only 30%, and o-ttp was not detectable. The concentrations of TnBP, TCEP, TPhP, TBEP and m-ttp in serum were ng g 1 lipid, ng g 1 lipid, < 4.2 ng g 1 lipid, < 49.9 ng g 1 lipid and < 23.1 ng g 1 lipid, respectively. Key Words: Organophosphate esters; Gel permeation chromatograph; Solid phase extraction; Human serum; Gas chromatography-mass spectrometer 1 Introduction Organophosphate esters (OPEs) are formed by substituting the hydrogen atom of phosphate with different substituent groups such as alkanes, aromatic hydrocarbons and halogenated hydrocarbons and so on [1]. Due to the highly flame retardancy and plasticity, OPEs are widely used in many products, such as varnish, polyurethane foam, upholstery and textiles. With the ban of commercial Penta-BDE, Octa-BDE, Deca-BDE and hexabromocyclodode- cane (HBCD) in Europe in , the production of OPEs is rapidly increasing all around the world. According to the data statistics, the production volumes of OPEs in China was tons, and the export was tons in 2007 [2]. Because OPEs were physically added to the materials, they could easily leach out into the environment during the produce, use and disposal process of materials. According to the toxicology studies, many OPEs showed the potential neurotoxic and carcinogenic, and inhibited the levels of hormones in human body [3]. Currently, OPEs were detected in Received 15 January 2015; accepted 25 April 2015 * Corresponding author. junjin3799@126.com This work was supported by the First-class University Construction Project of MinZu University of China (Nos. YLDX01013, 2015MDTD23C) and the Institution of Higher Education Innovation Talent Recruitment Program, China (No. B08044). Copyright 2015, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: /S (15)

2 the atmosphere, wastewater, surface water, groundwater, soil, indoor dust and many other environmental media in many countries [4 7]. OPEs were also detected in a variety of fishes, human urine, human blood, human milk, adipose tissue and other biota samples [8 10]. These results indicate that OPEs are ubiquitous in biota and abiotic. Thus, the determination of OPEs in human serum is very important to assess the influence of OPEs to human health. Currently, the main analytical methods for OPEs include high performance liquid chromatography-mass spectrometry (UPLC-MS), gas chromatography with nitrogen-phosphorus detector method (GC-NPD) and gas chromatography-mass spectrometry (GC-MS). For serum sample analysis, better qualitative and quantitative results can be obtained by using GC-EI-MS. The main extraction and purification methods for OPEs in human serum samples were hollow fiber extraction and microporous membranes extraction reported by Jonsson et al [11,12]. However, the method was very complex and circumscribed for a few kinds of OPEs. Shah et al [8] used solid phase micro-extraction combined with GC-ICP-MS and GC-TOF-MS to detect OPEs in human serum, but the instruments for GC-ICP-MS and GC-TOF-MS analysis were expensive and it also needed higher test environment. In the present study, an efficient method was established to analyze OPEs in human serum samples by gel permeation chromategraphy combined with solid phase extraction and GC-MS. 2 Experimental 2.1 Instruments and reagents Dichloromethane, acetone, methyl tert-butyl ether and hexane were of pesticide analysis grade (J.T. Baker, Phillipsburg, NJ, USA). Neutral alumina (100 μm) was purchased from Sigma-Aldrich (USA). The alumina were extracted with dichloromethane and activated (at 450 C for the alumina) for 4 h, then cooled and deactivated by adding 3% of the sorbent weight of deionized water. Silica gel ( μm) was purchased from Merck (Germany). The silica gel were extracted with dichloromethane, activated (at 130 C for the silica gel) for 12 h, then cooled and deactivated by adding 5% of the sorbent weight of deionized water. Gel Bio-beads SX-3 ( mm) was from Bio-Rad Laboratories, Richmond, CA. Anhydrous sodium sulfate was of analytical grade and was baked at 450 C for 4 h before use. Nitrogen (99.999%) and helium (99.999%) were purchased from Chengweixin (Beijing, China).Water was purified using a Milli-Q system (Millipore, Billerica, MA, USA). The standard TnBP, TCEP, TBEP, o-ttp, m-ttp and p-ttp were purchased from AccuStandard Corporation. The isotope-labeled OPE standards (d 12 -labeled TCEP and d 15 -labled TPhP) were from Cambridge Isotope Laboratories (Cambridge, MA, USA). The chemical structure and properties of OPEs are summarized in Table Sample preparation After 50 μl (total 50 ng) of d 12 -labeled TCEP and d 15 -labled TPhP standards with a concentration level of 1 ng μl 1 were added into 3 ml of serum sample, 6 ml of acetone and 4 ml of hexane/mtbe mixture (1:1, V/V) were added into the serum samples for the OPEs extraction. After rigorously mixing, the serum samples were centrifuged at 2500 rpm for 5 min and the organic phase were transferred into a glass test tube contained 4 ml of KCl (1%, w/w). Then the serum sample was extracted twice again with 3 ml of hexane/mtbe mixture (1:1, V/V) and the organic phase was integrated into the glass tube above. The collected organic phase was evaporated to dryness for the gravimetric determination of lipid content. After that, 4 ml of hexane and 2 ml of KOH (0.5 M) were added into the dryness sample. The sample was then mixed vigorously and centrifuged at 2500 rpm for 5 min, and the organic phase was collected into a test tube for the further analysis. And the aqueous phases were extracted twice with 3 ml of hexane and the organic phases were integrated into the tube above. Then the extractant above was concentrated to about 1 ml under a gentle nitrogen stream. After the macromolecular compound were removed by a gel permeation chromatography (26 g SX-3 GPC, 300 mm 25 mm), the extractant abovementioned was eluted with the mixed solution of hexane and dichloromethane (1:1, V/V), and the eluent was collected from 50 ml to 250 ml. Then the collected eluents were concentrated to 1 ml by rotary evaporation and purified by a silica/alumina composite column (the filters from top to bottom were 1.5 g of anhydrous sodium sulfate, 0.5 g of neutral silica gel, 0.1 g of alumina). The final OPEs were eluted from this column using 15 ml of acetone and concentrated to 100 μl under a gentle nitrogen stream for GC-MS analysis. 2.3 GC-MS conditions GC separation of OPEs was carried out on a DB-5MS chromatographic column (30 m 0.25 mm i.d., 0.1 μm film thickness, Agilent J&W GC Columns). The oven temperature was programmed as follows: the initial temperature was set at 100 C and held for 3 min, then ramped up to 300 C at a rate of 10 C min 1 and held for 3 min. Helium was used as carrier gas at a flow rate of 1.0 ml min 1. The injector temperature was 280 C. Samples were injected in splitless mode. The parameters of MS were as follows. Electron impact ion source (EI): the temperature of MS source was 230 C and the quadrupole temperature was 150 C; Positive chemical ionization source (PCI): the temperature of MS source was 230 C and the temperature of quadrupole was 150 C;

3 Table 1 Chemical structure and properties of organophosphate esters Names and abbreviations CAS number Structural formula Molecular structure Octanol-water partition coefficient (lgk ow ) Vapor pressure (mmhg at 25 C) Tri-n-butylphosphate, TnBP C 12 H 27 O 4 P Tri(2-chloroethyl)phosphate, TCEP C 6 H 12 Cl 3 O 4 P Triphenyl phosphate, TPhP C 18 H 15 O 4 P Tris(2-butoxyethyl) phosphate, TBEP C 18 H 39 O 7 P Tri-o-tolylphosphate, o-ttp C 21 H 21 O 4 P Tri-m-tolylphosphate, m-ttp C 21 H 21 O 4 P Tri-p-tolylphosphate, p-ttp C 21 H 21 O 4 P Electron capture negative ionization source (NCI): the temperature of MS source was 150 C and the temperature of quadrupole was 150 C. Moreover, select ion mode (SIM) was used as the monitoring mode. 3 Results and discussion 3.1 Optimization of lipid removal method Common lipid removal methods for serum samples are sulfuric acid, acidic silica gel and gel permeation chromatographic methods. Sulfuric acid and acidic silica gel could remove lipid from serum samples mainly because of their strong oxidation and dehydration, but sulfuric acid and acidic silica gel would strongly impact the recovery of OPEs. Gel permeation chromatography can not only effectively remove macromolecular lipids, pigments and proteins, but also ensure the structures of OPEs are not destroyed, thus achieving better recovery rate and reproducibility. In the present study, both the H 2 SO 4 -acidic silica gel and GPCsilica/alumina composite methods were compared. The results showed that the acidic silica gel method have poor recovery and reproducibility as a comparison with GPC-silica/alumina composite column method. In the lipid removal process by GPC-silica/alumina composite column method, the mixed standard samples of OPEs (including the labeled standards) were added to the top of GPC column and then eluted by 250 ml of hexane-dichloromethane (1:1, V/V). The eluent was collected into 25 tubes with 10 ml in each tube and then concentrated under a gentle nitrogen stream. As shown in Fig.1, for 26 g SX-3 GPC column, 50 ml of hexanedichloromethane (1:1, V/V) was first used to remove the main lipid, and then 150 ml of hexane-dichloromethane (1:1, V/V) was used to elute the OPEs from GPC. The experiment was operated three times parallelly, and the overall recoveries (mean ± standard deviation) for TnBP, d12-tcep, TCEP,

4 and acetone were compared. The experiment was carried out three times in parallel and the average recoveries of each OPEs were shown in Table 2. The results showed that acetone had the best elution efficiency, by which the recovery of all OPEs were more than 70.2%. 3.2 Optimization of MS source selection and condition Fig.1 Elution profiles of organophosphate esters from GPC with different elutes volume d15-tphp, TPhP, TBEP, o-ttp, m-ttp and p-ttp were 85.0% ± 12.4%, 83.3% ± 23.1%, 80.4% ± 5.4%, 99.1% ± 15.8%, % ± 2.3%, 97.0% ± 8.7%, 91.8% ± 6.9%, 92.3% ± 5.7% and 89.9% ± 6.1%, respectively. After GPC purification, there was still a small amount of small molecular impurities in the sample, which would interfere with the separation and quantification of the target compounds. Therefore, the sample should be further purified. Glass column packed with silica gel and alumina was commonly used for the purification of OPEs due to its better purification effect and non-damage to the OPEs structure. Silica/alumina composite column was filled from bottom to top with glass wool, 0.1 g of neutral alumina, 0.5 g of neutral silica gel and 1.5 g of anhydrous sodium sulfate. The eluent process was optimized by standard addition method. After the column was activated with 5 ml of hexane, 50 μl of 1 ng μl 1 mixed standard solution of OPEs was loaded into the column and then eluted with hexane, both the mixed solutions of hexane and dichloromethane (1:1, V/V), dichloromethane In the present study, we compared the sensitivity of electron impact ion source (EI), positive chemical ionization source (PCI) and electron capture negative ionization source (NCI). In the experiment, each OPEs were injected for the MS analysis according to the amount of 1 ng and 2 ng, respectively. In the selected ion mode, the most abundant ions in OPEs were selected to investigate the response of OPEs under the different sources and the results were shown in Table 3. As shown in Table 3, the response of OPEs were EI source > PCI source > NCI source, thus EI ionization source was selected to analyze OPEs. The prepared mixed standard solutions were then analyzed with EI source and selected ion mode (selected ions for each OPEs were shown in Table 4) and the total ion chromatograms of OPEs were shown in Fig Optimization of qualitative and quantitative ion The full scan mass spectra of standard OPEs obtained under the optimal GC-MS conditions are shown in Fig.3. According to the theory proposed by Ma et al [13], the splitting reaction of organic phosphate (TnBP, TCEP and TBEP) with substituent group of alkyl would occur under EI source condition by three times of Maxwell rearrangement, and the splitting process was [M] + [M R + 2H] + [M 2R + 3H] + [M 3R + 4H] + (R represent different substituents). Thus [M C 4 H 9 + 2H] + Table 2 Recovery of organophosphate esters using silica/alumina column (mean ± relative standard deviation) with different elutes Compounds n-hexane n-hexane/dichloromethane (1:1, V/V) Dichloromethane Acetone TnBP n.d ± ± ± 4.0 TCEP n.d. n.d ± ± 3.2 TPhP n.d ± ± ± 4.1 TBEP n.d. n.d ± ± 2.3 o-ttp n.d. n.d ± ± 1.6 m-ttp n.d. n.d ± ± 2.4 p-ttp n.d. n.d ± ± 1.5 *n.d., Not detected. Compound Electron impact ion source (EI) Table 3 Responses of organophosphate esters under different sources m/z Positive chemical ionization source (PCI) m/z Electron capture negative ionization source (ECNI) TnBP TCEP TPhP TBEP o-ttp m-ttp p-ttp m/z

5 Table 4 Retention time, qualitative and quantitative ions of organophosphate esters Analytes Retention time (min) Qualitative ion (m/z) Quantitative ion (m/z) TnBP , 155, d 12 -TCEP , 233, TCEP , 223, d 15 -TPhP , TPhP , TBEP , o-ttp , m-ttp , p-ttp , Fig.2 Total ion chromatograms of organophosphate esters and internal standard of d 12 -TCEP and d 15 -TPhP Fig.3 Mass spectra of seven kinds of organophosphate esters (m/z 211) fragment ion and [M 2(C 4 H 9 ) + 3H] + (m/z 155) were were selected as the characteristic ion for TnBP, the highest abundance [M-3 (C 4 H 9 ) + 4H] + (m/z 99) as TnBP quantitative ion. Similarly, [M C 6 H 13 O + 2H] + (m/z 299) and [M 2(C 6 H 13 O) + 3H] + (m/z 199) were selected as TBEP characteristic ions, and the higher abundance [M-C 6 H 13 O + 2H] + as its quantitative ions. Compared with TnBP and TBEP, due to its strong electronegative Cl substituents, TCEP produced a large number of m/z 249 [M Cl] + fragment ion and m/z 63 [ClCH 2 CH 2 ] + fragment ion in the process of ionization. This was mainly because the substitutions of Cl at the γ-h position would result in a low abundance of rearrangement of [M C 2 H 4 Cl + 2H] + (m/z 223) and [M 2(C 2 H 4 Cl) + 3H] + (m/z 161). Thus the ions of [M C 2 H 4 Cl + 2H] + (m/z 223) and [M 2(C 2 H 4 Cl) + 3H H 2 O] + (m/z 143) with a higher abundance were selected as TCEP qualitative ions, and [M Cl] + (m/z 249) with the highest abundance was selected as TCEP quantitative ions. The most abundant peak for aryl-substituted organic phosphate esters such as o-ttp, m-ttp, p-ttp and TPhP were all the molecular ion peak [M] + under EI condition, thus the most abundant m/z 368 ion was selected as the quantitative ion for the MS analyses of o-ttp, m-ttp, p-ttp, and m/z 326 ion as the quantification ion for TPhP MS analysis. According to

6 the literature reported by Xin et al [14], TPhP could choose a relatively high abundance of m/z 170 peak as its qualitative, and o-ttp, m-ttp, p-ttp could choose m/z 165 ion peak as their qualitative peak. But through the analysis of its full scan spectra, we found that the fragment at the spectrum of m/z 170 for TPhP was [C 12 H 10 O] + which was formed by the recombination of fragment ions dissociated from [M C 6 H 5 ] + and [M OC 6 H 5 ] +. Therefore, it cannot reflect the structure of TPhP using [C 12 H 10 O] + as the qualitative ion for TPhP. In the experiment, the optimal [M OC 6 H 5 ] + (m/z 233) and [M O 2 C 6 H 7 ] + (m/z 215) were chosen as characteristic qualifier ion. Similarly, m/z 165 and m/z 179 also could not reflect o-ttp, m-ttp, p-ttp structural information, thus [M C 7 H 7 ] + (m/z 277) and [C 7 H 7 ] + (m/z 91) were selected as o-ttp, m-ttp, p-ttp qualitative ions. After the optimization of qualifier ions, the structures of rganic phosphate esters could be accurately identified by GC-MS. 3.4 Calibration curve and detection limit The prepared series standard solutions were analyzed under the optimal conditions with 1 ng μl 1 d 12 -TCEP as internal standard for TnBP and TCEP and 1 ng μl 1 d 15 -TPhP as internal standard for TPhP, TBEP, o-ttp, m-ttp and p-ttp. Calibration curve were plotted on the basis of the concentration ratios and peak area ratios. The regression equations, correlation coefficient and linear range of organophosphate esters were shown in Table 5. The limits of detection (LOD) were ng g 1 lipid weight (S/N = 3) and limits of quantitation (LOQ) were 2 45 ng g 1 lipid weight (S/N = 10). 3.5 Recovery and precision After the mixed standard solutions of OPEs (50 μl 1 ng μl 1 ) were added into deionized water (n = 3) and the mixed standard solutions of d 12 -TCEP and d 15 -TPhP (50 μl 1 ng μl 1 ) were added into the human serum samples, the standard solutions were pretreated according to the method described in Section 2.2 and then detected by GC-MS. The recoveries of each substance in the deionized water samples were all above 75% and in human serum samples, the average recovery of d 12 -TCEP and d 15 -TPhP were 86.3% ± 21.6% and 103.1% ± 16.5%, respectively, with a good reproducibility. Meanwhile, three blank samples were prepared and detected, and no OPEs were found in the three blank samples, showing there was no contamination during the experiment. The results indicated that the recoveries met the requirement of analysis with a good reproducibility. 3.6 Application to human serum samples 9 human serum samples and 3 blank samples were prepared and detected by the method proposed here. The detection rates were more than 90% for TnBP, TCEP, TPhP, TBEP and m-ttp and 30% for p-ttp, and no o-ttp was detected in all samples. Meanwhile, no OPEs were detected in blank samples. The concentrations of TnBP, TCEP, TPhP, TBEP and m-ttp were ng g 1 lipid, ng g 1 lipid, < 4.2 ng g 1 lipid, < 49.9 ng g 1 lipid and < 23.1 ng g 1 lipid, respectively. The detection rate of m-ttp was the highest among the three isomers of TTP. However, the most harmful isomers o-ttp was not detected in human serum samples. The concentration of TCEP was an order of magnitude higher than other OPEs, indicating the pollution of TCEP in human serum was seriously, thus we should strengthen the monitor of the environmental behavior of TCEP. In conclusion, the method of gel permeation chromatography-solid phase extraction-gas chromatography/ mass spectrometry can be used to detect OPEs with high recovery and good reproducibility in human serum samples, gel permeation chromatography can be used to remove lipid effectively without damaging the structure of OPEs, and solid phase extraction can be used to effectively reduce the interference and achieve good separation effect. The method is effective for the detection of OPEs in human serum samples with good reproducibility and quality control. Table 5 Regression equations, correlation coefficient and linear range of organophosphate esters Compound Linear range (μg L 1 ) Regression equations Correlation coefficient TnBP Y = x TCEP Y = x TPhP Y = x TBEP Y = 16.13x o-ttp Y = x m-ttp Y = x p-ttp Y = x References [1] Wang X W, Liu J F, Yin Y G. Process. Chem., 2010, 22(10): [2] European Flame Retardants Association. efra.com/content, 2009 [3] van der Veen I, de Boer J. Chemosphere, 2012, 88(10): [4] Wang X W, He Y Q, Lin L, Zeng F, Luan T G. Sci. Total Environ., 2014, 470:

7 [5] Lu J X, Ji W, Ma S T, Yu Z Q, Wang Z, Li H, Ren G F, Fu J M. Chinese J. Anal. Chem., 2014, 42(6): [6] Cao S, Zeng X, Song H, Li H, Yu Z, Sheng G, Fu J. Environ. Toxicol. Chem., 2012, 31(7): [7] Bergh C, Torgrip R, Emenius G, Ostman C. Indoor Air, 2011, 21(1): [8] Shah M, Meija J, Cabovska B, Caruso J A. J. Chromatogr. A, 2006, 1103(2): [9] van den Eede N, Neels H, Jorens P G, Covaci A. J. Chromatogr. A, 2013, 1303: [10] Kim J W, Isobe T, Muto M, Tue N M, Katsura K, Malarvannan G, Sudaryanto A, Chang K H, Prudente M, Viet P H, Takahashi S, Tanabe S. Chemosphere, 2014, 116: [11] Jonsson O B, Nilsson U L. Anal. Bioanal. Chem., 2003, 377(1): [12] Jonsson O B, Nilsson U L. J. Sep. Sci., 2003, 26(9-10): [13] Ma Y, Hites R A. J. Mass Spectrom., 2013, 48(8): [14] Xing Y N, Wang X, Chen Z Y, Suo Y Y, Lin H X. Chinese J. Anal. Chem., 2012, 40(9):